The present invention relates to methods for the prognostic and diagnostic of neurodegenerative disease, kits related to such methods and methods to identify candidate compounds for preventing and treating neurodegenerative disease.
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by the progressive degeneration of motor neurons in the brain and spinal cord. Approximately 10% of ALS cases are familial and 90% are sporadic. Recently, TAR DNA binding protein 43 (TDP-43) has been implicated in ALS1. TDP-43 is a DNA/RNA-binding 43 kDa protein that contains a N-terminal domain, two RNA recognition motifs (RRMs) and a glycine-rich C-terminal domain, characteristic of the heterogeneous nuclear ribonucleoprotein (hnRNP) class of proteins2. TDP-43, normally observed in the nucleus, is detected in pathological inclusions in the cytoplasm and nucleus of both neurons and glial cells of ALS and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) cases1, 3. The inclusions consist prominently of TDP-43 C-terminal fragments (CTFs) of ˜25 kDa. The involvement of TDP-43 with ALS cases led to the discovery of TDP-43 mutations found in ALS patients. Dominant mutations in TARDBP, which codes for TDP-43, were reported by several groups as a primary cause of ALS4-9 and may account for ˜3% of familial ALS cases and ˜1.5% of sporadic cases.
Neuronal overexpression at high levels of wild-type or mutant TDP-43 in transgenic mice caused a dose-dependent degeneration of cortical and spinal motor neurons but with no cytoplasmic TDP-43 aggregates10-13, raising up the possibility that an upregulation of TDP-43 in the nucleus rather than TDP-43 cytoplasmic aggregates may contribute to neurodegeneration. The physiological role of TDP-43 and the pathogenic pathways of TDP-43 abnormalities are not well understood. TDP-43 is essential for embryogenesis14 and postnatal deletion of the TDP-43 gene in mice caused downregulation of Tbc1d1, a gene that alters body fat metabolism15. Proteins known to interact with TDP-43 have also been implicated in protein refolding or proteasomal degradation including ubiquitin, proteasome-beta subunits, SUMO-2/3 and Hsp7016.
Because TDP-43 is ubiquitously expressed and several studies have supported the importance of glial cells in mediating motor neuron injury17-19, additional proteins which might interact with TDP-43 in LPS-stimulated microglial (BV-2) cells were searched. The rationale for choosing microglial BV-2 cells was that TDP-43 deregulation may occur not only in neurons but also in microglial cells. Moreover, there are recent reports of increased levels of LPS in the blood of ALS patients20 and of an upregulation of LPS/TLR-4 signaling associated genes in peripheral blood monocytes from ALS patients21. Accordingly, the search was biased for proteins interacting with TDP-43 when microglia are activated by LPS. Surprisingly, co-immunoprecipitation assays and mass spectrometry led us to identify the p65 subunit of NF-κB as a binding partner of TDP-43. Furthermore, the results show that TDP-43 mRNA was abnormally upregulated in the spinal cord of ALS subjects. These results reported here led to further explore the physiological significance of the interaction between TDP-43 and p65 NF-κB.
As the symptoms of ALS are similar to those of other neuromuscular disorders, many of which are treatable, ALS is difficult to diagnose. The diagnosis is usually based on a complete neurological examination and clinical tests.
There is therefore a need for methods for evaluating a subject predisposed to developing a neurodegenerative disease such as ALS and FTLD-U or suffering from these neurodegenerative diseases as well as method to identify new candidate compounds useful for the prevention and/or treatment of neurodegenerative diseases.
The present inventors have surprisingly found an interaction between TDP-43 and p65 NF-κB in subjects suffering from a neurodegenerative disease. The present inventors have also found that levels of TDP-43 and p65 mRNA are elevated in subjects suffering from a neurodegenerative disease.
The present invention relates to methods measuring or evaluating interaction between TDP-43 and p65 for diagnosis, prognosis, monitoring the progression of the disease or for identifying drug candidates.
The present invention also relates to measuring the level of TDP-43 and/or p65 mRNA.
Kits for measuring the interaction between TDP-43 and p65 and for measuring the levels of TDP-43 and p65 mRNA are also provided by the present invention.
The present invention also relates to the use of the interaction level between TDP-43 and p65 as a biochemical marker for monitoring the progression or the regression of a neurological disease.
The present invention also relates to a method for the diagnostic of a subject predisposed or suspected of developing a neurodegenerative disease or suffering from a neurodegenerative disease, the method comprising the step of:                determining the level of interaction between a TDP-43 polypeptide or fragment thereof and a p65 polypeptide or fragment thereof in a biological sample of the subject,wherein observing an elevated level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof in the biological sample relative to a reference level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates that the subject is predisposed or suspected of developing a neurodegenerative disease or is suffering from a neurodegenerative disease.        
The present invention also relates to a method for the diagnostic of a subject predisposed or suspected of developing a neurodegenerative disease or suffering from a neurodegenerative disease, the method comprising the steps of:                contacting a TDP-43 polypeptide or fragment thereof with a TDP-43 agent in a biological sample of the subject;        contacting a p65 polypeptide or fragment thereof with a p65 agent in the biological sample; and        detecting the TDP-43 agent and/or the p65 agent to determine the level of interaction between the TDP-43 polypeptide or fragment thereof and the p65 polypeptide or fragment thereof,wherein detecting an elevated level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof in the biological sample relative to a reference level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates that the subject is predisposed or suspected of developing a neurodegenerative disease or is suffering from a neurodegenerative disease.        
The present invention also relates to a method for the diagnostic of a subject predisposed or suspected of developing a neurodegenerative disease or suffering from a neurodegenerative disease, the method comprising the step of:                determining the level of TDP-43 mRNA in a biological sample of the subject, wherein observing an elevated level of TDP-43 mRNA in the biological sample relative to the reference level of TDP-43 mRNA indicates that the subject is predisposed or suspected of developing a neurodegenerative disease or is suffering from a neurodegenerative disease.        
The present invention also relates to a method for the diagnostic of a subject predisposed or suspected of developing a neurodegenerative disease or suffering from a neurodegenerative disease, the method comprising the steps of:                isolating TDP-43 mRNA from a biological sample of a subject; and        detecting the level of TDP-43 mRNA in the biological sample of the subject,wherein detecting an elevated level of TDP-43 mRNA in the biological sample relative to the reference level of TDP-43 mRNA indicates that the subject is predisposed or suspected of developing a neurodegenerative disease or is suffering from a neurodegenerative disease.        
The present invention also relates to a kit for the diagnostic of a subject predisposed to or suspected of developing a neurodegenerative disease or suffering from a neurodegenerative disease, the kit comprising:                i) at least one TDP-43 specific antibody or fragment thereof;        ii) at least one p65 specific antibody or fragment thereof;        iii) a reference corresponding to the level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof,        iv) a container, and        v) a buffer or an appropriate reagent.        
The present invention also relates to a kit for the diagnostic of a subject predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease, the kit comprising:                i) at least one set of specific primers for determining the level of TDP-43 mRNA;        ii) a reference corresponding to the level of TDP-43 mRNA,        iii) a container, and        iv) a buffer or an appropriate reagent.        
The present invention also relates to a method for identifying a candidate compound useful for preventing and/or treating a neurodegenerative disease, the method comprising the steps of:                a) contacting the candidate compound with a biological system comprising TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof,        b) measuring the ability of the candidate compound to modulate the activation of NF-κB p65 in the biological system, and        c) determining if the candidate compound is useful for preventing and/or treating a neurodegenerative disease based on the result of step b).        
The present invention also relates to a method for identifying a candidate compound useful for preventing and/or treating a neurodegenerative disease, the method comprising the steps of:                a) contacting the candidate compound with a biological system comprising TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof        b) measuring the ability of the candidate compound to reduce or inhibit the interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof, and        c) determining if the candidate compound is useful for preventing and/or treating a neurodegenerative disease based on the result of step a).        
The present invention also relates to a method for monitoring the progression or the regression of a neurodegenerative disease in a subject, the method comprising the step of:                determining the level of interaction between a TDP-43 polypeptide or fragment thereof and a p65 polypeptide or fragment thereof in a biological sample of the subject,wherein observing an increased level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates a progression of the neurodegenerative disease and wherein observing a decreased level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates a regression of the neurodegenerative disease.        
The present invention also relates to a method for monitoring the progression or the regression of a neurodegenerative disease in a subject, the method comprising the steps of:                contacting a TDP-43 polypeptide or fragment thereof with a TDP-43 agent in a biological sample of the subject;        contacting a p65 polypeptide or fragment thereof with a p65 agent in the biological sample; and        detecting the TDP-43 agent and/or the p65 agent to determine the level of interaction between the TDP-43 polypeptide or fragment thereof and the p65 polypeptide or fragment thereof;wherein detecting an increased level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates a progression of the neurodegenerative disease and wherein observing a decreased level of interaction between TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof indicates a regression of the neurodegenerative disease.        
The present invention also relates to a use of the interaction level between a TDP-43 polypeptide or fragment thereof and p65 polypeptide or fragment thereof in a biological sample as a biochemical marker for monitoring the progression or the regression of a neurodegenerative disease in a subject.
The present invention also relates to a use of at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof for treating a subject suffering from a neurodegenerative disease.
The present invention also relates to a use of a pharmaceutical composition comprising at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier for treating a subject suffering from a neurodegenerative disease.
The present invention also relates to use of at least one withanolide compound or pharmaceutically acceptable salt thereof for treating a subject suffering from a neurodegenerative disease.
The present invention also relates to a use of a pharmaceutical composition comprising at least one withanolide compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier for treating a subject suffering from a neurodegenerative disease.
The present invention also relates to a method for treating a subject suffering from a neurodegenerative disease comprising the step of administering at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof to the subject.
The present invention also relates to a method for treating a subject suffering from a neurodegenerative disease comprising the step of administering a pharmaceutical composition comprising at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable salt thereof to the subject.
The present invention also relates to a method for treating a subject suffering from a neurodegenerative disease comprising the step of administering at least one withanolide compound or pharmaceutically acceptable salt thereof to the subject.
The present invention also relates to a method for treating a subject suffering from a neurodegenerative disease comprising the step of administering a pharmaceutical composition comprising at least one withanolide compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier to the subject.
The present invention also relates to a non-human transgenic animal model of neurodegenerative disease, wherein the genome of the non-human transgenic model comprises a human TDP-43 genomic fragment operably linked to a human TDP-43 promoter and wherein the non-human transgenic model expresses human TDP-43 polypeptide in a moderate level.
The present invention also relates to an expression cassette comprising the sequence of TDP-43WT, TDP-43A315T or TDP-43G348C.
The present invention also relates to a transgenic cell transformed with the expression cassette as defined herein.
The present invention also relates to a method for identifying or confirming the utility of a candidate compound useful for preventing and/or treating a neurodegenerative disease, the method comprising the steps of:                a) administering the candidate compound to the non-human transgenic model as defined herein;        b) measuring the effect of the candidate compound on the non-human transgenic model in behavioral task test or by in vivo bioluminescence imaging; and        c) determining if the candidate compound is useful for preventing and/or treating the neurodegenerative disease based on the result of step c).        
The term “subject” refers to any subject susceptible of suffering or suffering from neurodegenerative disease. Specifically, such a subject may be, but not limited to, human, an animal (e.g. cat, dog, cow, horse, etc.). More specifically, the subject consists of a human.
The terms “predisposed” and “suspected” refer to a subject who does not yet experience or display the pathology or symptomatology of the disease but who may has increased probability or increased risk of developing neurodegenerative disease.
The expression “neurodegenerative disease” refers to the progressive loss of structure or function of neurons such as ALS, frontotemporal lobar degeneration, Alzheimer, motor neuron disease or Parkinson. Neurodegenerative disease also relates to disease in which TDP-43 is involved. In one embodiment, the neurodegenerative disease is associated with TDP-43 proteinopathy.
TDP-43 polypeptides as well as polynucleotides are well known in the art. For example see NM_007375.3, Gene Bank AK222754.1 or UniProt Q13148 Representative polypeptide sequence (SEQ ID NO:1) and polynucleotide sequence (SEQ ID NO: 2).
In one aspect, TDP-43 may comprise 2 RRM domains (RNA Recognition Motif) (amino acids 106-176 and 191-262 as shown in the examples), a NLS (Nuclear Localization Signal) domain, a N-terminal domain (amino acids 1-105) and a C-terminal domain (amino acids 274-414).
In one embodiment, TDP-43 polypeptide includes a sequence at least 65% to 95% identical, at least 65%, 70%, 75%, 80%, 85%, 90% identical or at least 95% identical to part or all of the sequence shown in SEQ ID NO. 1 or fragment thereof.
In one embodiment, TDP-43 polynucleotide includes a sequence at least 65% to 95% identical, at least 65%, 70%, 75%, 80%, 85%, 90% identical or at least 95% identical to part or all of the sequence shown in SEQ ID NO. 2 or fragment thereof.
p65 polypeptides as well as polynucleotides are well known in the art. For example see M62399.1. Representative polypeptide sequence (SEQ ID NO:3) and polynucleotide sequence (SEQ ID NO:4).
In another aspect, p65 may comprise RRM1 (amino acids 104-200), RRM2 (amino acids 191-262) and Glycine rich domains (amino acids 275-413) as well as a NLS domain (amino acids 82-98).
In another embodiment, p65 polypeptide includes a sequence at least 65% to 95% identical, at least 65%, 70%, 75%, 80%, 85%, 90% identical or at least 95% identical to part or all of the sequence shown in SEQ ID NO. 3 or fragment thereof.
In one embodiment, p65 polynucleotide includes a sequence at least 65% to 95% identical, at least 65%, 70%, 75%, 80%, 85%, 90% identical or at least 95% identical to part or all of the sequence shown in SEQ ID No. 4 or fragment thereof.
Techniques for determining nucleic acid and amino acid “sequence identity” are also known in the art. Typically, such techniques include determining the nucleotide sequence of the mRNA for a gene and/or determining the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. In general, “identity” refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity.” The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the “BestFit” utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). Another method of establishing percent identity which can be used in the context of the present invention is the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects “sequence identity.” Other suitable programs for calculating the percent identity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR.
The term “polypeptide or fragments thereof” as used herein refers to peptides, oligopeptides and proteins. This term also does not exclude post-expression modification of polypeptides. For example, polypeptides that include the covalent attachment of glycosyl groups, acetyl groups, lipid groups and the like are encompassed by the term polypeptide. The term “fragment thereof”, as used herein, refers to polypeptide that may comprise for example 50%, 60%, 70%, 80%, 90%, 95% or more of the polypeptide sequence of the full length reference polypeptide. In one aspect the fragment is a fragment that is functional (e.g. retains the activity of the complete polypeptide or polynucleotide)
TDP-43 polypeptide functional fragments that interact with p65 include TDP-43 fragments spanning the RRM-I domain (amino acids 98-176) and/or the N-terminal domain (amino acids 31-81) of TDP-43 polypeptide.
The term “antibody” is intended herein to encompass monoclonal antibody and polyclonal antibody.
The term “mRNA” refers to mRNA or cDNA sequence of more than one nucleotide in either single or duplex form. The mRNA in accordance with the invention may be isolated by any known method. TDP-43 mRNA and p65 mRNA refer to mRNA sequences encoding TDP-43 and p65 polypeptides, respectively.
As used herein, the term “sample” refers to a variety of sample types obtained from a subject and can be used in a diagnostic assay. The definition encompasses blood, urine, cerebrospinal fluid and other liquid samples of biological origin. The definition also encompass solid tissue samples such as a biopsy of specimen or tissue culture or cells derived therefrom such as cortical neurons, microglial cells, myeloid cells or spinal cord extract.
As used herein, the interaction of TDP-43 with p65 refers to an interaction sufficient to activate NF-κB p65 pathway. The interaction may be for example, ionic, non-covalent or covalent binding of TDP-43 with p65. The level of interaction between TDP-43 and p65 may be detected and quantified within the biological sample. The detection of TDP-43 interacting with p65 may involve a detecting agent, which may be for instance, a specific antibody such as a purified monoclonal or polyclonal antibody raised against TDP-43. In such a case, the determination of interaction between TDP-43 and p65 is achieved by contacting a TDP-43 specific antibody with the biological sample under suitable conditions. As known in the art a second detecting agent, which may be, for instance a specific antibody such as a purified monoclonal or polyclonal antibody raised against p65 is needed to measure the interaction between TDP-43 and p65 within the biological sample. In such a case, the determination of interaction between TDP-43 and p65 is achieved by contacting a p65 specific antibody with TDP-43-antibody complex under suitable conditions to obtain a TDP-43-antibody-p65-antibody complex. The determination of interaction between TDP-43 and p65 in the biological sample may also be performed by contacting a p65 specific antibody with the biological sample under suitable conditions prior to contacting the biological sample with a TDP-43 specific antibody. Techniques for determining or measuring interaction between polypeptides are well known in the art and may include for example SDS-PAGE, ELISA, immunoprecipitation, co-immunoprecipitation, Western Blot assay, immunostaining, EMSA supershift or radioimmunoassay.
In one aspect, p65 interacts with the N-terminal portion of TDP-43. In another aspect, p65 polypeptide interacts with one of the RRM domain of TDP-43 such as RRM domain of amino acids 106-176.
As used herein, the expression “reference level” of a given polypeptide or polynucleotide refers to a level of polypeptide or polynucleotide present in a healthy subject i.e. not suffering from a neurodegenerative disease or as the case may be the level of the subject at different points for evaluating the progression of the disease.
In accordance with this invention, an elevated level of interaction between TDP-43 and p65 is indicative of the subject's risk of being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease or as the case may be is indicative of the progression of the disease in the subject. When the level of interaction between TDP-43 and p65 in the subject to be tested and the level of interaction between TDP-43 and p65 in a healthy subject are substantially identical, the subject's risk of being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease may be low. When the difference in the levels of interaction between TDP-43 and p65 in the subject to be tested and the level of interaction between TDP-43 and p65 in healthy subject increases, the risk of the subject being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease also increases. For example, an elevated level of interaction between TDP-43 and p65 could be at least 1.8 fold higher than the reference level.
In accordance with this invention, an elevated level of TDP-43 and/or p65 mRNA is indicative of the subject's risk of being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease or as the case may be is indicative of the progression of the disease in the subject. When the levels of TDP-43 and/or p65 mRNA in the subject to be tested and the level of TDP-43 and/or p65 mRNA in a healthy subject are substantially identical, the subject's risk of being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease is low. When the difference in the levels of TDP-43 and/or p65 mRNA in the subject to be tested and the level of TDP-43 and/or p65 mRNA in the healthy subject is increased, the risk of being predisposed to developing a neurodegenerative disease or suffering from a neurodegenerative disease is also increased. For example an elevated level of TDP-43 mRNA could be at least 2.5 folds higher than the reference level and the elevated level of p65 mRNA could be at least 4-folds higher than the reference level.
In accordance with this invention, the level of mRNA in the biological sample can be determined by methods well known in the art, for example by PCR or hybridization assays. Primers used for determining the level of TDP-43 mRNA may be the nucleic acid sequences set forth in SEQ ID NOs. 5 and 6 (SEQ ID NO. 5: GCGGGAAAAGTAAAAGATGTC, SEQ ID NO. 6: ATTCCTGCAGCCCGGGGGATCC) and primers used for determining the level of p65 mRNA may be the nucleic acid sequences set forth in SEQ ID NOs. 7 and 8 (SEQ ID NO. 7: GAGCGACTGGGGTTGAGAAGC, SEQ ID NO. 8: CCCATAGGCACTGTCTTCTTTCACC).
As used herein, the expressions “TDP-43-specific antibody” and “p65-specific antibody” refer to antibodies that bind to one or more epitopes of TDP-43 or p65 respectively, but which do not substantially recognize and bind to other molecules in a sample containing a mixed population of antigenic molecules.
The term “primer” is used herein to denote a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. As known in the art, a primer is used as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase, or reverse transcriptase.
The expression “TDP-43 specific primers” or “p65 specific primer” refers to primers that bind to a TDP-43 cDNA or p65 cDNA, respectively but which do not substantially recognize and/or bind to other molecules in a sample containing a mixed population of polynucleotide sequences.
The present invention further provides kits for use with the diagnostic methods of the present invention. Such kits typically comprise two or more components necessary for performing a diagnostic assay. Components may be compounds, appropriate reagents, containers, buffer and/or equipment. For example, one container within a kit may contain at least two specific antibodies wherein one antibody specifically binds to TDP-43 and the other antibody specifically binds to p65 as described herein. One or more additional containers may enclose elements, such as reagents or buffers, to be used in the assay.
Alternatively, a kit may be designed to detect the level of mRNA or cDNA encoded by TDP-43 polypeptide in a biological sample. Such kits generally comprise at least one set of oligonucleotide primers, as described herein, that hybridizes to a polynucleotide encoding TDP-43 polypeptide. Such an oligonucleotide may be used, for example, within a PCR or hybridization assay. Additional components that may be present within such kits include a reagent or container to facilitate the detection or quantification of mRNA encoding TDP-43 polypeptide.
In another aspect, the kit designed to detect the level of mRNA or cDNA encoded by TDP-43 polypeptide may further comprise a second set of oligonucleotide primers that hybridizes to cDNA encoding p65 polypeptide, as described herein.
In another aspect of the present invention, there is provided a method of identifying a candidate compound to determine whether the compound is useful for preventing or treating neurodegenerative diseases. The observation that TDP-43 interacts with p65 (e.g. as a co-activator) in subject strongly indicates a role for NF-κB signaling in neurodegenerative disease as shown herein. Therefore compounds capable of modulating, preventing or reducing activation of NF-κB p65 may be useful in preventing or treating neurodegenerative disease. The methods of the present invention are also useful for screening libraries of compounds in order to identify compounds that may be used as compounds for preventing or treating neurodegenerative disease.
The expression “candidate compound” includes compounds such as small molecules, nucleic acids, antibodies or polypeptides capable of interacting with a biological target molecule, in particular with a protein, in such a way as to modify the biological activity thereof. The expression includes compounds capable of interacting with TDP-43 or p65 in such a way that the interaction between TDP-43 and p65 is modified. In one aspect the compounds are capable of reducing or inhibiting the activation of NF-κB p65.
The expression “biological system” refers to a suitable biological assay or biological model. The biological assay can be an in vitro assay wherein the interaction between p65 and TDP-43 is measured, or the activation of NF-κB p65 is measured. The biological model can be any suitable model allowing the evaluation of the interaction between p65 and TDP-43 or the activation of NF-κB p65. The model can be an organism that has been modified in order to over-express TDP-43 and/or p65. In one embodiment, the model is TDP-43 transgenic mouse. In one embodiment, the TDP-43 transgenic mouse is the transgenic mouse described herein. In another embodiment, the model can be any cell types wherein NF-κB p65 is activated (translocated to the nucleus).
The ability of the compound to modulate, reduce and/or inhibit the activation of NF-κB p65 can be measured by method well known in the art such as ELISA assay, immunoprecipitation assay, coimmunoprecipitation assay, Western Blot assay, immunostaining or radioimmunoassay. NF-κB is known to be involved in pro-inflammatory and innate immune response. Therefore, level of gene activation such as TNF-α, Il-1β, IL-6, or NADPH oxidase 2 could be assessed in order to determine whether or not the candidate compound modulates, reduces and/or inhibits activation of NF-κB p65. Techniques to assess level of gene activation are well known in the art such as reporter gene assays.
In another aspect of the present invention, there is provided a method for monitoring the progression or the regression of a neurodegenerative disease. A higher level of interaction between TDP-43 and p65 over time indicates that the disease progresses whereas a lower level of interaction between TDP-43 and p65 over time indicates that the disease regresses. Monitoring the level of interaction between TDP-43 and p65, over time may be useful in clinical screening wherein a compound is tested on a subject. Therefore, the ability of a compound to modulate, reduce and/or inhibit the activation of NF-κB p65 in a subject can be monitored over a desired period.
Monitoring the level of interaction between TDP-43 and p65 over time can be measured by method well known in the art and as described herein. The interaction levels between TDP-43 and p65 in samples can be monitored during a desired period. For example, a sample can be obtained from a subject at different time such as hourly, daily, weekly, monthly or yearly and the interaction levels between TDP-43 and p65 are determined for each different time. In another embodiment, the level of mRNA of TDP-43 and/or p65 can be monitored during a desired period to determine the progression or the regression of the disease.
Another aspect of the present invention is to provide the use of the interaction level between TDP-43 and p65 as a biochemical marker. The term “biochemical marker” is known to the person skilled in the art. In particular, biochemical markers are gene expression products which are differentially expressed, i.e., upregulated or downregulated, in presence or absence of a certain disease. A biochemical marker can be a protein or peptide and can be for example the level of interaction between TDP-43 and p65 and/or the mRNA level of TDP-43 and/or p65. The level of a biochemical can indicate the presence or absence of the disease and thus allow diagnosis. The biochemical marker can then be used to monitor the progression or the regression of a disease over a desired period.
In one aspect of the present invention, there is provided use of at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier for treating a subject suffering from a neurodegenerative disease.
In another aspect, there is provided a method for treating a subject suffering from a neurodegenerative disease. The method comprises the step of administering at least one TDP-43 interacting compound or a pharmaceutically acceptable salt thereof or administering a pharmaceutical composition comprising at least one TDP-43 interacting compound or a pharmaceutically acceptable salt and a pharmaceutical acceptable salt thereof to the subject.
The expression “TDP-43 interacting compound” includes compounds such as small molecules, nucleic acids, antibodies or polypeptides capable of interacting with TDP-43 such that the activation of p65 NFκB pathway is reduced or inhibited. The interaction may be for example electrostatic interactions, dipolar interactions, entropic effects or dispersion forces. In one embodiment, the compound may interact with the RRM1 and/or RRM2 domain (amino acids 106-176 and 191-262) of TDP-43 polypeptide. The level of interaction between TDP-43 and the compound may be detected and quantified by known methods such as ELISA, radioimmunoassay, immunoprecipitation assay, Western blot assay, immunostaining assay, EMSA assay, EMSA super shift assay, Chromatin Immunoprecipitation Assay, DNA Pull-down Assay, Microplate Capture and Detection Assay, Reporter Assay or AlphaScreen technology63.
In one aspect, the compound is a nucleic acid molecule. The expression “nucleic acid molecule” is intended to include DNA molecule (e.g. cDNA or genomic DNA) and RNA molecules (e.g. mRNA). The nucleic acid molecule can be single-stranded or double-stranded. The nucleic acid molecule can be genomic DNA or can be synthesized by known techniques.
In another aspect of the present invention, the nucleic acid molecules comprise the following single-stranded DNA molecules disclosed by Cassel et al.65: TG12 (TGTGTGTGTGTGTGTGTGTGTGTG) (SEQ ID NO:21), TG8 (TGTGTGTGTGTGTGTG) (SEQ ID NO:22), TAR-32 (CTGCTTTTTGCCTGTACTGGGTCTCTGTGGTT) (SEQ ID NO: 23), TG6 (TGTGTGTGTGTG) (SEQ ID NO:24), TG4 (TGTGTGTG) (SEQ ID NO:25) or dAC12 (ACACACACACACACACACACACAC) (SEQ ID NO:26) and the following double-stranded RNA molecules also disclosed by Cassel et al65: UG12 (UGUGUGUGUGUGUGUGUGUGUGUG) (SEQ ID NO:27), UG8 (UGUGUGUGUGUGUGUG) (SEQ ID NO:28), UG6 (UGUGUGUGUGUG) (SEQ ID NO:29), UCUU3 (UCUUUCUUUCUU) (SEQ ID NO:30) and rAC12 (ACACACACACACACACACACACAC) (SEQ ID NO:31).
The expression “treating a subject” refers to treatment that halts the progression of, reduces the pathological manifestations of, or entirely eliminates a condition in a patient. Following TDP-43 polypeptide interaction with the nucleic acid molecule, TDP-43 is less likely to interact with p65 thus reducing p65 NFκB activation. As shown herein by reducing p65 NFκB activation, the motor impairment is ameliorated in a subject suffering from a neurodegenerative disease. For instance, the motor impairment can be improved by at least 4% compared to the untreated subject. Methods for qualification and quantification of a reduction in pathological manifestations in a subject suffering from neurodegenerative disease are known in the art such as rotarod performance test, motor control test, postural evoked response, adaptation test or balance strategy analysis, barnes maze task and step-through passive avoidance test.
Nucleic acid molecule can be administered to the subject in an encapsulated form such as liposome, virus, nanocapsule or microsphere as known in the art. Methods for encapsulating nucleic acid molecule are also known in the art.
Another aspect of the invention provides the use of at least one withanolide compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one withanolide compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier for treating a subject suffering from a neurodegenerative disease.
In another aspect, there is provided a method for treating a subject suffering from a neurodegenerative disease. The method comprises the step of administering at least one withanolide compound or a pharmaceutically acceptable salt thereof or administering a pharmaceutical composition comprising at least one withanolide compound or a pharmaceutically acceptable salt thereof and a pharmaceutical acceptable carrier to the subject.
The term “at least one withanolide compound” refers to steroidal compounds with an ergosterol skeleton in which C-22 and C-26 are oxidized to form a δ-lactone. Withanolide can be isolated from Withania somnifera or another Withania species. Withanolide can also be semi-synthetically produced from withanolide natural products or can be produced by total synthesis. Examples of known withanolide are: withaferin A, withanolide N, withanolide O, withanolide D, withanolide E, withanolide P, withanolide S, withanolide Q, withanolide R, withanolide G, withanolide H, withanolide I, withanolide J, withanolide K, withanolide U, withanolide Y, analogs or pharmaceutically salt thereof.
In one aspect, the withanolide is withaferin A, an analog, or a pharmaceutically acceptable salt thereof.
The term “analog” includes analogs of withaferin A described in WO2010/053655 and WO2010/030395.
As described herein, withanolide compound such as withaferin-A-treated TDP-43WT or TDP-43G348C transgenic mice show an ameliorated motor impairment of at least 4% compared to their untreated TDP-43WT or TDP-43G348C transgenic mice. Motor behavior can be analysed with known techniques such as rotarod performance test, motor control test, postural evoked response, adaptation test or balance strategy analysis, barnes maze task and step-through passive avoidance test.
As used herein, the term “pharmaceutical composition” refers to the combination of an active agent (e.g., withanolide) with a carrier, inert or active, making the composition especially suitable for therapeutic use.
It is noted in that the present invention is intended to encompass all pharmaceutically acceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) of the compounds, regardless of whether such ionized forms and solvates are specified since it is well known in the art to administer pharmaceutical agents in an ionized or solvated form. It is also noted that unless a particular stereochemistry is specified, recitation of a compound is intended to encompass all possible stereoisomers (e.g., enantiomers or diastereomers depending on the number of chiral centers), independent of whether the compound is present as an individual isomer or a mixture of isomers.
The expression “pharmaceutically acceptable salts” are meant those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicylic, succinic, toleune p sulphonic, tartaric, acetic, trifluoroacetic, citric, methanesulphonic, formic, benzoic, malonic, naphthalene 2 sulphonic and benzenesulphonic acids. Salts derived from amino acids are also included (e.g. L-arginine, L-Lysine). Salts derived from appropriate bases include alkali metals (e.g. sodium, lithium, potassium) and alkaline earth metals (e.g. calcium, magnesium).
With regards to pharmaceutically acceptable salts, see also the list of FDA approved commercially marketed salts listed in Table I of Berge et al., Pharmaceutical Salts, J. of Phar. Sci., vol. 66, no. 1, January 1977, pp. 1-19.
It will be appreciated by those skilled in the art compounds can exist in different polymorphic forms. As known in the art, polymorphism is an ability of a compound to crystallize as more than one distinct crystalline or “polymorphic” species. A polymorph is a solid crystalline phase of a compound with at least two different arrangements or polymorphic forms of that compound molecule in the solid state. Polymorphic forms of any given compound are defined by the same chemical formula or composition and are as distinct in chemical structure as crystalline structures of two different chemical compounds.
It will be appreciated that the amount of compounds required for use in treatment will vary not only with the particular compound selected but also with the route of administration, the nature of the condition for which treatment is required and the age and condition of the patient and will be ultimately at the discretion of the attendant physician.
The desired dose may conveniently be presented in a single dose or as divided dose administered at appropriate intervals, for example as two, three, four or more doses per day. While it is possible that, for use in therapy, the compounds may be administered as the raw chemical it is preferable to present the active ingredient as a pharmaceutical composition. The invention thus further provides a pharmaceutical combination or composition of the compounds as described herein or a pharmaceutically acceptable salt thereof together with one or more pharmaceutically acceptable carriers therefore and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
Pharmaceutical compositions include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), transdermal, vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation. The compositions may, where appropriate, be conveniently presented in discrete dosage units and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association the active with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired composition.
Pharmaceutical compositions suitable for oral administration may conveniently be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution, a suspension or as an emulsion. The active ingredient may also be presented as a bolus, electuary or paste. Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, disintegrants, or wetting agents. The tablets may be coated according to methods well known in the art. Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.
The compounds may also be formulated for parenteral administration (e.g., by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilization from solution, for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.
For topical administration to the epidermis, the compounds may be formulated as ointments, creams or lotions, or as a transdermal patch. Such transdermal patches may contain penetration enhancers such as linalool, carvacrol, thymol, citral, menthol and t-anethole. Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents. Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilizing agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
Compositions suitable for topical administration in the mouth include lozenges comprising active ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
Pharmaceutical compositions suitable for rectal administration wherein the carrier is a solid are for example presented as unit dose suppositories. Suitable carriers include cocoa butter and other materials commonly used in the art, and the suppositories may be conveniently formed by admixture of the active compound with the softened or melted carrier(s) followed by chilling and shaping in moulds.
Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
For intra-nasal administration the compounds or combinations may be used as a liquid spray or dispersible powder or in the form of drops. Drops may be formulated with an aqueous or non-aqueous base also comprising one more dispersing agents, solubilizing agents or suspending agents. Liquid sprays are conveniently delivered from pressurized packs.
For administration by inhalation the compounds or combinations are conveniently delivered from an insufflator, nebulizer or a pressurized pack or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount.
Alternatively, for administration by inhalation or insufflation, the compounds or combinations may take the form of a dry powder composition, for example a powder mix of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or e.g. gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator.
As used herein, the expression “an acceptable carrier” means a vehicle for the combinations and compounds described herein that can be administered to a subject without adverse effects. Suitable carriers known in the art include, but are not limited to, gold particles, sterile water, saline, glucose, dextrose, or buffered solutions. Carriers may include auxiliary agents including, but not limited to, diluents, stabilizers (i.e., sugars and amino acids), preservatives, wetting agents, emulsifying agents, pH buffering agents, viscosity enhancing additives, colors and the like.
It will be appreciated that the amount of a compound required for use in treatment will vary not only with the particular compound selected but also with the route of administration, the nature of the condition for which treatment is required and the age and condition of the patient and will be ultimately at the discretion of the attendant physician. In general however a suitable dose will be in the range of from about 0.001 to about 100 mg/kg of body weight per day, for example, in the range of 0.01 to 50 mg/kg/day, or, for example, in the range of 0.1 to 40 mg/kg/day. The compound is conveniently administered in unit dosage form; for example containing 1 to 2000 mg, 10 to 1500 mg, conveniently 20 to 1000 mg, most conveniently 50 to 700 mg of active ingredient per unit dosage form.
In another embodiment of the present invention, dosages may be estimated based on the results of experimental models, optionally in combination with the results of assays of the present invention. Generally, daily oral doses of active compounds will be from about 0.01 mg/kg per day to 2000 mg/kg per day. Oral doses in the range of 10 to 500 mg/kg, in one or several administrations per day, may yield suitable results. In the event that the response of a particular subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are also contemplated in some cases to achieve appropriate systemic levels of the composition
In another aspect, the present invention provides a non-human transgenic animal model suffering from a neurodegenerative disease which can be used as a model for testing therapeutic approaches.
The expression “non-human transgenic animal model” refers to an animal whose genetic material has been altered. In one embodiment, the genome of the animal has been altered to introduce therein a DNA sequence such as human TDP-43 polynucleotide. Methods for creating transgenic animal are known in the art such as DNA microinjection, embryonic stem cell-mediated gene transfer and retrovirus-mediated gene transfer.
The animal model can be a rodent, rat, sheep, monkey, goat, mouse, cat, dog or pig. In one embodiment, the animal model is a mouse.
The term “genome” as used herein refers to an organism's hereditary information. The genome includes both the genes and the non-coding sequences of the DNA/RNA molecule.
The expression “human TDP-43 genomic fragment operably linked to a human TDP-43 promoter” refers to a TDP-43 nucleic acid sequence fragment comprising TDP-43 coding sequence, the introns, the 3′ sequence autoregulating TDP-43 synthesis as described by Polymenidou et al 2011 and the human TDP-43 promoter as described in Swamp et al. Brain, 2011, 134, p. 2610-2626. The genomic fragment can be obtained from a library composed of genomic fragments. In one embodiment the TDP-43 genomic fragment linked to its endogenous promoter is obtained from a human bacterial artificial chromosome clone. In another embodiment, the human TDP-43 promoter is ligated upstream of the TDP-43 genomic fragment. Thus the expression of TDP-43 polypeptide is driven by its endogenous promoter. The TDP-43 genomic fragment comprises the 3′ auto-regulating TDP-43 synthesis sequence within an alternatively spliced intron in the 3′UTR of the TDP-43 pre mRNA. Methods for obtaining genomic fragments, generating libraries, ligating promoters to nucleic acid sequences are known in the art such as molecular cloning.
The expression “expresses human TDP-43 polypeptide in a moderate level” refers to the animal model expressing human TDP-43 at a level that allows the animal to develop signs of neurological dysfunction. For instance, neurological sign of dysfunction can include increasing TDP-43 polypeptide or mRNA expression, ubiquitinated TDP-43 inclusion, transactive response TDP-43 cleavage fragments, intermediate filament abnormalities, axonopathy, neuroinflammation, memory capabilities, impaired learning and memory capabilities or motor dysfunction. In one embodiment, the animal model develops the neurological dysfunction at about 10 months of age. In one embodiment, the RNA expression level of the human TDP-43 in the transgenic animal model is about 3 fold higher as compared with the RNA level of the animal endogenous TDP-43. Methods for quantifying RNA level expression are known in the art such as quantitative real time PCR or northern blot.
In another embodiment, the human TDP-43 genomic fragment operably linked to the human TDP-43 promoter comprises TDP-43WT sequence isolated from clone RPCI-11, number 829B14, TDP-43A315T sequence having a known mutation at position 315, or TDP-43G348C sequence having a known mutation at position 348 as described in Swamp et al Brain, 2011, 134, p. 2610-2626. The sequences comprising the mutations (TDP-43A315T and TDP-43G348C) can be derived from a human genome as mentioned above or the mutations can be inserted within the TDP-43 wild type sequence using site-directed mutagenesis as known in the art.
The expression “amyotrophic lateral sclerosis” is used herein to refer to any neurodegenerative disease that usually attacks both upper and lower motor neurons and causes degeneration throughout the brain and spinal cord.
The expression “frontotemporal lobar degeneration disease” refers to a group of disorders associated with atrophy in the frontal and temporal lobes. Frontotemporal lobar degeneration disease (FTLD) can include FTLD-tau characterized by tau inclusion, FTLD-TDP43 characterized by ubiquitin and TDP-43 inclusion (FTLD-U), FTLD-FUS characterized by FUS cytoplasmic inclusions and dementia lacking distinctive histology (DLDH).
The expression “TDP-43 proteinopathy” refers to neurodegenerative disease associated with the accumulation and/or aggregation of abnormal or misfolded TDP-43 polypeptide.
In another embodiment, the present invention provides an expression cassette comprising the sequence of TDP-43WT, TDP-43A315T or TDP-43G348C as described above.
The expression “expression cassette” as used herein refers to the combination of promoter elements with other transcriptional and translational regulatory control elements which are operably linked. A heterologous gene sequence can be inserted into the expression cassette for the purpose of expression of said gene sequence. The expression cassette is capable of directing transcription which results in the production of an mRNA for the desired gene product. The expression cassette is inserted into a plasmid to produce an expression vector. Such an expression vector directs expression of the heterologous protein in host cells.
In another embodiment, there is provided a transgenic cell transformed with the expression cassette as described above. The term “transformed” refers to the DNA-mediated transformation of cells referring to the introduction of the expression cassette DNA into the cells.
In one embodiment, the transgenic cell is obtained from a mouse.
In another embodiment, the present invention provides a method for identifying or confirming whether a compound candidate is useful for preventing and/or treating a neurodegenerative disease. The candidate compound is administered to the non-human transgenic model as defined herein. The effect of the compound on the non-transgenic model is measured by assessing a behavioral task test or by in vivo bioluminescence imaging. When the non-human transgenic model shows an improved behavioral task test or a decrease of neurological dysfunction observed by in vivo bioluminescence it strongly indicates that the candidate compound is useful for preventing or treating the neurodegenerative disease.
The expression “behavioral task test” refers to an experimental task which assesses the capacity of an organism to process environmental cues and respond accordingly. For instance, spatial learning, memory, motor skill, balance, coordination or physical condition of the organism can be measured. Methods for measuring behavioral task are known in the art such as Barnes maze task test, Morris water navigation task, Radial arm task, step-through passive avoidance test and accelerating rotorod test.
In one embodiment, the behavioral task test is the Barnes maze task which refers to a test for measuring spatial learning and memory. The test is described in (Prut et al., 2007).
In a further embodiment, the behavioral task test is the step-through passive avoidance test which refers to an aversive conditioning paradigm in which the subject learns to associate a particular context with the occurrence of an aversive event. For instance, passive avoidance behavior in rodents is the suppression of the innate preference for the dark compartment of the test apparatus following exposure to an inescapable shock.
In another embodiment, the behavioral task test is the accelerating rotorod test which refers to a test for measuring riding time, endurance, balance or coordination. In the test, a subject is placed on a horizontally oriented, rotating cylinder (rod) suspended above a cage floor, which is low enough not to injure the animal, but high enough to induce avoidance of fall. Subjects naturally try to stay on the rotating cylinder, or rotarod, and avoid falling to the ground. The length of time that a given animal stays on this rotating rod is a measure of their balance, coordination, physical condition, and motor-planning. The speed of the rotarod is mechanically driven, and may either be held constant, or accelerated.
The expression <<in vivo bioluminescence imaging>> as used herein refers to the process of light emission in living organism. For instance, 11C-PiB PET could be used to assess change in fibrillar amyloid-beta load in vivo. As also known, luciferase can be used to assess the progression and/or the regression of neurological dysfunction in vivo.
From the data presented here, it is proposed that a TDP-43 deregulation in ALS may contribute to pathogenic pathways through abnormal activation of p65 NF-κB. Several lines of evidence support this scheme: (i) proof of a direct interaction between TDP-43 and p65 NF-κB was provided by immunoprecipitation experiments using protein extracts from cultured cells, from TDP-43 transgenic mice and from human ALS spinal cord samples, (ii) reporter gene transcription assays and gel shift experiments demonstrated that TDP-43 was acting as co-activator of p65 NF-κB through binding of its N-terminal domain to p65, (iii) the levels of mRNAs for both TDP-43 and p65 NF-κB were substantially elevated in the spinal cord of ALS subjects as compared to non-ALS subjects whereas immunofluorescence microscopy of ALS spinal cord samples revealed an abnormal nuclear localization p65 NF-κB, (iv) cell transfection studies demonstrated that an overexpression of TDP-43 can provoke hyperactive innate immune responses with ensuing enhanced toxicity on neuronal cells whereas in neurons TDP-43 overexpression increased their vulnerability to toxic environment, (v) in vivo treatment of TDP-43 transgenic mice with an inhibitor of NF-κB reduced inflammation and ameliorated motor deficits.
This is the first report of an upregulation of mRNAs encoding TDP-43 in post-mortem frozen spinal cords of sporadic ALS. A recent study has provided evidence of increased TDP-43 immuno-detection in the skin of ALS patients38 but it failed to demonstrate whether this was due to upregulation in TDP-43 mRNA expression. The process that underlies a 2.5-fold increase in TDP-43 mRNA levels in ALS, whether it is transcriptional or mRNA stability remains to be investigated. It seems unlikely that copy number variants could explain an increase of TDP-43 gene transcription as variations in copy number of TARDBP have not been detected in cohorts of ALS39-41. Actually, the pathogenic pathways of TDP-43 abnormalities in ALS are not well understood. To date, much attention has been focused of cytoplasmic C-terminal TDP-43 fragments that can elicit toxicity in cell culture systems42-45. However, it is noteworthy that neuronal overexpression at high levels of wild-type or mutant TDP-43 in transgenic mice caused a dose-dependent degeneration of cortical and spinal motor neurons but without massive cytoplasmic TDP-43 aggregates10. This suggests that an upregulation of TDP-43 in the nucleus rather than TDP-43 cytoplasmic aggregates may contribute to neurodegeneration in these mouse models. As shown here, an overexpression of TDP-43 can trigger pathogenic pathways via NF-κB activation.
The transcription factor NF-κB is a key regulator of hundreds of genes involved in innate immunity, cell survival and inflammation. Since the nuclear translocation and DNA binding of NF-κB are not sufficient for gene induction46, 47, it has been suggested that interactions with other protein molecules through the transactivation domain48-50 as well as its modification by phosphorylation51 might play a critical role. It has been reported that transcriptional activation of NF-κB requires multiple co-activator proteins including CREB-binding protein (CBP)/p30048,49, CBP-associated factor, and steroid receptor coactivator 152. These coactivators have histone acetyltransferase activity to modify the chromatin structure and also provide molecular bridges to the basal transcriptional machinery. NF-κB p65 was also found to interact specifically with Fused in Sarcoma (FUS) protein, another DNA/RNA binding protein which is involved in ALS53-55.
The results revealed robust effects of TDP-43 on the activation of NF-κB and innate immune responses. After transfection with TDP-43 species, microglial cells challenged with LPS exhibited much higher mRNA levels for pro-inflammatory cytokines, Nox-2 and NF-κB mRNA when compared to untransfected cells after LPS stimulation. TDP-43 overexpression makes microglia hyperactive to immune stimulation resulting in enhanced toxicity toward neighbouring neuronal cells with involvement of reactive oxygen species (ROS) and increased nitrite levels (NO). Moreover, the adverse effects of TDP-43 upregulation are not limited to microglial cells. Primary cortical neurons overexpressing TDP-43 transgenes by ˜3-fold exhibited increased susceptibility to the toxic effects of excess glutamate or LPS-activated microglia (FIG. 13A).
The presence of ALS-linked mutations in TDP-43 (A315T or G348C) did not affect the binding and activation of p65 NF-κB. This is not surprising because the deletion mutant analysis revealed that a region spanning part of the N-terminal domain and RRM1 of TDP-43 is responsible for interaction with p65 whereas most TDP-43 mutations in ALS occur in the C-terminal domain, which is dispensable for p65 NF-κB activation (FIG. 4). In fact, the cytotoxicity assays with primary cells from TDP-43 transgenic mice revealed that, at similar levels of mRNA expression, the adverse effects of mutant TDP-43 were more pronounced than TDP-43wt. These results could be explained by the observation that ALS-linked mutations in TDP-43 increase its protein stability56. From the data presented here, we propose the involvement in ALS of a pathogenic pathway due to nuclear increase in TDP-43 levels (FIG. 6). Recent TDP-43 studies with Drosophila suggested that the TDP-43 toxicity may occur in absence of inclusions formation and that neurotoxicity requires TDP-43 RNA-binding domain57. These results are consistent with the model presented here of TDP-43 toxicity and with data demonstrating interaction of TDP-43 with p65 via the RNA recognition motif RMM1.
The finding that TDP-43 acts as co-activator of p65 suggests a key role for NF-κB signalling in ALS pathogenesis. This is corroborated by the abnormal 4-fold increase of p65 NF-κB mRNA in the spinal cord of human ALS (FIG. 6) and by the nuclear localization of p65 (FIG. 1L-N; FIG. 2). Remarkably, an overexpression of TDP-43 species by ˜3-fold in transgenic mice, at levels similar to the human ALS situation (2.5-fold), was sufficient to cause during aging nuclear translocation of p65 NF-κB in the spinal cord (FIG. 1F-H). It should be noted that TDP-43 itself does not cause NF-kB activation (FIG. 7) and that it does not upregulate p65. It seems that a second hit is required. For example, LPS or other inducers such as pathogen-associated molecular patterns can trigger through TLR signalling p65 NF-kB nuclear localization. Cytokines such as TNF and IL-1 can also trigger p65 activation. In ALS, the second hit(s) triggering innate immune responses remain to be identified. There is recent evidence for involvement of LPS in ALS20, 21 and of endogenous retrovirus (HEVR-K) expression58. Here we show that aging is associated with p65 nuclear translocation in the spinal cord of TDP-43 transgenic mice (FIG. 12) but the exact factors underlying this phenomenon remain to be defined.
There is a recent report of mutations in the gene coding for vasolin-containing protein (VCP) associated with 1-2% familial ALS cases59. It is well established that VCP is involved in the control of the NF-kB pathway through regulation of ubiquitin-dependent degradation of IκB-α. For instance, mutant VCP expression in mice resulted in increased TDP-43 levels and hyper-activation of NF-κB signalling60, 61. Moreover, some ALS-linked mutations have been discovered in the gene coding for optineurin, a protein which activates the suppressor of NF-κB62, further supporting a convergent NF-κB-pathogenic pathway. Thus, the data presented in here as well as ALS-linked mutations in the VCP and optineurin genes59, 61, 62 are all supporting a convergent NF-κB pathogenic pathway in ALS. The present invention shows that inhibitors of NF-κB activation are able to attenuate the vulnerability of cultured neurons overexpressing TDP-43 species to glutamate-induced or microglia-mediated toxicity. Moreover, pharmacological inhibition of NF-κB by WA treatment attenuated disease phenotypes in TDP-43 transgenic mice. From these results, it is proposed that NF-κB signalling should be considered as potential therapeutic target in ALS treatment (FIG. 16).
We report here the generation and characterization of novel transgenic mouse models of ALS-FTLD based on expression of genomic fragments encoding TDP-43 WT or mutants (A315T and G348C). The mouse models reported here carry TDP-43 transgenes under its own promoter resulting in ubiquitous and moderate expression (˜3 fold) of hTDP-43 mRNA species. Most of the mouse models of TDP-43 reported previously have shown early paralysis followed by death. However, these mouse models are based on high expression levels of TDP-43 transgenes that can mask age-dependent pathogenic pathways. Mice expressing either wild-type or mutant TDP-43 (A315T and M337V) showed aggressive paralysis accompanied by increased ubiquitination (Wegorzewska et al., 2009; Stallings et al., 2010; Wils et al., 2010; Xu et al., 2010) but the lack of ubiquitinated TDP-43 positive inclusions raises concerns about their validity as models of human ALS disease. Another concern is the restricted expression of TDP-43 species with the use of Thy1.2 and Prion promoters. To better mimic the ubiquitous and moderate levels of TDP-43 occurring in the human context, it seems more appropriate to generate transgenic mice with genomic DNA fragments of TDP-43 gene including its own promoter. As in human neurodegenerative disease, our TDP-43 transgenic mice exhibited age-related phenotypic defects including impairment in contextual learning/memory and spatial learning/memory as determined by passive avoidance test and Barnes maze test. Long term memory of 10-months old TDP-43G348C transgenic mice was severely impaired according to Barnes maze test. The TDP-43G348C, TDP-43A315T and to a lesser extent TDP-43Wt mice exhibited also motor deficits as depicted by significant reductions in latency in the accelerating rotarod test.
Cognitive and motor deficits in TDP-43 transgenic mice prompted us to test the underlying pathological and biochemical changes in these mice. Western blot analysis of spinal cord lysates of transgenic mice revealed ˜25-kDa and ˜35-kDa TDP-43 cleavage fragments which increased in levels with age. Previous studies demonstrated cytotoxicity of the ˜25-kDa fragment (Zhang et al., 2009). Immunofluorescence studies with human TDP-43 specific monoclonal antibodies revealed TDP-43 cytoplasmic aggregates in the spinal cord of TDP-43G348C, TDP-43A315T and to lesser extent in TDP-43Wt mice. The cytoplasmic TDP-43 positive inclusions were ubiquitinated. The TDP-43 positive ubiquitinated cytoplasmic inclusions along with ˜25-kDa cytotoxic fragments are reminiscent of those described in studies on ALS and FTLD-U patients (Neumann et al., 2006). The co-immunoprecipitation of ubiquitin with anti-TDP-43 antibody and inversely of TDP-43 with anti-ubiquitin antibody (FIG. 18U&V) using spinal cord samples from TDP-43G348C mice further confirmed the association of TDP-43 with ubiquitinated protein aggregates. However, TDP-43 itself was not extensively ubiquitinated. A thorough survey of articles on TDP-43 led us to the conclusion that there is no compelling biochemical evidence in literature supporting the general belief that TDP-43 is the major poly-ubiquitinated protein in the TDP-43 positive inclusions. We could find only one blot from one ALS case in one paper (Neumann et al., 2006) that revealed a very weak detection of high molecular weight smear with anti-TDP-43 after TDP-43 immunoprecipitation. A subsequent paper by (Sanelli et al., 2007) has concluded from 3D-deconvolution imaging that TDP-43 is not in fact the major ubiquitinated target in ubiquitinated inclusions of ALS.
The TDP-43 transgenic mice described here exhibit perikaryal and axonal aggregates of intermediate filaments, another hallmark of degenerating motor neurons in ALS (Carpenter, 1968; Corbo and Hays, 1992; Migheli et al., 1993). Before the onset of behavioural changes in these mice, there is formation of peripherin aggregates in the spinal cord and brain sections of TDP-43G348C as well as in TDP-43A315T transgenic mice. These peripherin inclusions were also seen in the hippocampal region of the brain of TDP-43G348C mice. Normally peripherin is not expressed in brain. However, it is known that peripherin expression in the brain can be upregulated after injury or stroke (Beaulieu et al., 2002). The enhanced peripherin levels in these mice are probably due to an upregulation of IL-6, a cytokine that can trigger peripherin expression (Sterneck et al., 1996). Sustained peripherin overexpression by over 4 fold in transgenic mice was found previously to provoke progressive motor neuron degeneration during aging (Beaulieu et al., 1999). In addition, we detected in TDP-43 transgenic mice the presence of abnormal splicing variants of peripherin, such as Per 61, that can contribute to formation of IF aggregates (Robertson et al., 2003). Using Per61 specific antibodies we detected peripherin inclusions in the spinal cord sections of TDP-43G348C mice, but not in TDP-43Wt mice (FIG. 19). The occurrence of specific splicing peripherin variants has also been reported in human ALS cases (Xiao et al., 2008).
In addition we detected neurofilament protein anomalies in TDP-43G348C mice. Double immunofluorescence revealed the detection of neurofilament NF-H and NF-M in inclusion bodies with peripherin in the spinal cord of TDP-43G348C mice. Moreover, we found that neurofilament NF-L is downregulated in the spinal cord lysates of TDP-43G348C mice, a phenomenon which has also been observed in motor neurons of ALS cases (Wong et al., 2000). A decrease in NF-L levels may explain in part the age-related axonal atrophy detected in TDP-43 mice. Previous studies with NF-L knockout mice demonstrated that such substantial shift in calibres of large myelinated axons provokes a reduction of axon conduction velocity by ˜3 fold (Kriz et al., 2000). In large animals with long peripheral nerves this would cause neurological disease. A loss of neurofilaments due to a homozygous recessive mutation in the NEFL gene was found recently to cause a severe early-onset axonal neuropathy (Yum et al., 2009).
Age-related neuroinflammation constitutes another striking feature of the TDP-43 transgenic mice. In vivo imaging of biophotonic doubly transgenic mice bearing TDP-43 and GFAP-luc transgenes showed that astrocytes are activated as early as 20 weeks in the brain of GFAP-luc/TDP-43G348C mice followed by activation in the spinal cord at ˜30 weeks of age. The signal intensity for astrocytosis in GFAP-luc/TDP-43A315T and GFAP-luc/TDP-43Wt was less than in GFAP-luc/TDP-43G348C mice. It is noteworthy that the induction of astrogliosis detected in the brain and spinal cord in all three TDP-43 mouse models preceded by 6 to 8 weeks the appearance of cognitive and motor defects. This finding is in line with the recent view of an involvement of reactive astrocytes in ALS pathogenesis (Barbeito et al., 2004; Di Giorgio et al., 2007; Julien, 2007; Nagai et al., 2007; Di Giorgio et al., 2008).
In conclusion, the TDP-43 transgenic mice described here mimic several aspects of the behavioural, pathological and biochemical features of human ALS/FTLD including age-related development of motor and cognitive dysfunction, cytoplasmic TDP-43 positive ubiquitinated inclusions, intermediate filament abnormalities, axonopathy and neuroinflammation. Why there is no overt degeneration in our TDP-43 mouse models? Unlike previous TDP-43 transgenic mice, these transgenics were made with genomic fragment that contains 3′ sequence autoregulating TDP-43 synthesis (Polymenidou et al., 2011). So, the TDP-43 levels remain moderate. The ubiquitous TDP-43 overexpression by about 3 folds in these mice mimics the 2.5-fold increase of TDP-43 mRNA measured in the spinal cord of human sporadic ALS by quantitative real-time PCR (our unpublished result). In human ALS cases carrying TDP-43 mutations, it takes many decades before ALS disease onset. The factors that trigger the onset are unknown but perhaps future studies with TDP-43 mouse models might provide some insights. In any case, our new TDP-43 mouse models should provide valuable tools for unraveling pathogenic pathways of ALS/FTLD and for preclinical drug testing.
The present invention will be more readily understood by referring to the following examples. These examples are illustrative of the wide range of applicability of the present invention and are not intended to limit its scope. Modifications and variations can be made therein without departing from the spirit and scope of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred methods and materials are described. The issued patents, published patent applications, and references that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail.